The estrogen receptor (ER) occupies central roles in normal mammary gland development and breast cancer signaling by regulating gene expression and reshaping the epigenetic state of nearby chromatin. Natural endocrine modulations such as time of menarche, pregnancy, and menopause can modify the risk of breast cancer, and chemopreventive ER-targeting interventions are effective in reducing breast cancer incidence. However, the mechanism behind the altered function of this steroid receptor transcription factor during tumorigenesis remains unknown. Estrogen stimulation of ER+ breast cancer cells has been observed to induce chromatin remodeling and histone modifications, and this dysregulation of epigenetic networks may be a potential mechanism of breast tumorigenesis. ER also interacts with other proteins since women possessing germline BRCA1 and BRCA2 mutations display a predilection for different breast cancer subtypes with BRCA1 mutation carriers developing predominantly ER- cancers and BRCA2 carriers developing ER+ cancers, while prophylactic oophorectomy paradoxically decreases the risk of both types of BRCA1/2-associated cancers. The proposed Specific Aims will investigate the role of ER in normal mammary differentiation and breast tumorigenesis by utilizing epithelial cells isolated from primary breast tumors and phenotypically normal tissue via fluorescence-activated cell sorting (FACS) of dissociated single cell suspensions. Aim 1 will compare estrogen signaling between normal mammary tissue and ER+ breast tumors by RNA-seq expression profiling and chromatin immunoprecipitation and sequencing (ChIP-seq) for ER to identify estrogen-responsive genes and genomic ER binding sites for validation of implicated genes and cooperating transcription factors. Aim 2 will then seek to explain the epigenetic changes during normal mammary differentiation and breast tumorigenesis by utilizing histone modification ChIP-seq and DNAse I hypersensitivity sequencing (DHS-seq) to describe distinct epigenetic and chromatin accessibility programs. Aim 3 will use the tumor subtype selectivity of BRCA1 and BRCA2-associated cancers to characterize various tumorigenic mechanisms through RNA-seq expression profiling, ER and histone mark ChIP-seq along with DHS-seq on cells isolated from BRCA1/2 mutation carriers undergoing prophylactic mastectomies. Unraveling the ER signaling program in the normal breast of these various genotypes may hold the key to understanding the mechanisms behind these clinical observations of risk and prevention. Distinguishing resulting gene expression from the differential interplay between transcriptional regulation and epigenetics is ultimately critical to understanding breast cancer risk and innovating preventive measures.